Volume 16, Issue 2, Pages 304-313 (July 2016) Genetic Isolation of Hypothalamic Neurons that Regulate Context-Specific Male Social Behavior  Marta E. Soden, Samara M. Miller, Lauren M. Burgeno, Paul E.M. Phillips, Thomas S. Hnasko, Larry S. Zweifel  Cell Reports  Volume 16, Issue 2, Pages 304-313 (July 2016) DOI: 10.1016/j.celrep.2016.05.067 Copyright © 2016 The Authors Terms and Conditions

Cell Reports 2016 16, 304-313DOI: (10.1016/j.celrep.2016.05.067) Copyright © 2016 The Authors Terms and Conditions

Figure 1 Molecular Profile and Connectivity of PMV-DAT Neurons (A) Schematic generation of DAT-Cre-RiboTag mice. (B) Atlas image depicting the PMV; boxed region is depicted in immunohistochemistry image of Rpl22-HA (right) and inset. Scale bar, 250 μm. (C) Cartoon depicting RiboTag technique: in Cre-positive cells the ribosomal protein Rpl22 is labeled with an HA tag; immunoprecipitation of HA isolates ribosome-associated mRNAs. (D) Fold enrichment (IP compared to input) of specific mRNAs isolated from PMV-DAT neurons (n = 3 pooled samples, each from 2–3 mice). (E) Projections of PMV-DAT neurons are revealed by expression of synapto-EGFP in axon terminals. Atlas images show approximate distance from bregma; indicated brain regions are color-coded. See also Figure S1. Cell Reports 2016 16, 304-313DOI: (10.1016/j.celrep.2016.05.067) Copyright © 2016 The Authors Terms and Conditions

Figure 2 PMV-DAT Neurons Are Principally Glutamatergic and Do Not Release Dopamine (A and B) Example traces (A) and current/voltage plots (B) showing light-evoked dopamine release detected using FSCV in control slices expressing ChR2 in VTA-DAT neurons, recording in the NAc. No signal was detected in the VMHVL when ChR2 was expressed in PMV-DAT neurons, even with pre-loading of L-DOPA. (C) Example traces showing that PMV-DAT neurons did not spontaneously fire in slice but did fire accommodating action potentials with current injection (top trace), while VTA-DAT neurons did fire spontaneously (bottom trace). (D and E) Example traces (D) and quantification (E) of Ih current in PMV-DAT neurons (top) and VTA-DAT neurons (bottom). (F and G) Example traces (F) and quantification (G) of SK currents in PMV-DAT neurons (gray) and VTA-DAT neurons (black). (H) DIC (top) and fluorescent (bottom) images of an acute slice with fluorescent ChR2-mCherry fibers in the VMHVL and patch electrode visible. Scale bar, 250 μm. (I) Example trace of EPSC in the VMHVL evoked by 5-ms blue light stimulation; the EPSC could be blocked by bath application of CNQX (10 μM). (J) Top: example recordings showing a light-evoked monosynaptic EPSC (black trace, holding at −60 mV) and a delayed, unsynchronized IPSC (gray trace, holding at 0 mV). Bottom: example recording showing a light-evoked monosynaptic EPSC (initial fast depolarization) and a delayed, unsynchronized EPSC. All traces are averages of 15 sweeps. (K) Proportion of VMHVL neurons recorded that received each type of synaptic input. See also Figure S2. Cell Reports 2016 16, 304-313DOI: (10.1016/j.celrep.2016.05.067) Copyright © 2016 The Authors Terms and Conditions

Figure 3 PMV-DAT Neurons Regulate Intruder-Specific Behavior (A and B) Images (A) and quantification (B) of Fos levels in PMV-DAT neurons during social encounters (n = 3 mice/group; one-way ANOVA F(5,12) = 12.26, p < 0.001; Tukey’s multiple comparison ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; scale bar, 100 μm). (C) Social investigation of resident animal by intruder (experimental) was decreased following inhibition of PMV-DAT neurons by CNO/hM4Di (n = 10–11 mice/group; two-way repeated-measures ANOVA virus × CNO F(1,19) = 4.43, p < 0.05; Bonferroni multiple comparisons ∗p < 0.05). (D and E) Inhibition of PMV-DAT neurons did not affect the investigation of a male (D) or female (E) intruder by a resident (experimental). (F) Inhibition of PMV-DAT neurons by CNO/hM4Di did not affect preference for a mouse over an object. (G) Inhibition of PMV-DAT neurons eliminated the preference for a novel mouse versus a familiar mouse; n = 10–11 mice/group; two-way repeated-measures ANOVA: F(1,19) = 7.38, p < 0.05; Bonferroni multiple comparisons: ∗p < 0.05). Data are represented as mean ± SEM. See also Figure S3. Cell Reports 2016 16, 304-313DOI: (10.1016/j.celrep.2016.05.067) Copyright © 2016 The Authors Terms and Conditions

Figure 4 PMV-DAT Neurons Regulate Social Behaviors through Glutamate Release (A) Atlas and histology images showing ChR2-mCherry expression in the PMV; dashed lines indicate track mark from fiber optic. (B) Schematic of cohabitation behavioral assay. (C) Activation of PMV-DAT neurons increased social investigation of a familiar cage mate (n = 6–8 animals/group; Student’s t test ∗p < 0.05). (D) Activation did not increase investigation of a familiar object. (E) Pairing one side of a two-chambered box with light stimulation did not lead to a significant preference for either side. (F) Inactivation of vGlut2 in DAT-vGlut2 KO mice eliminates light-evoked excitatory currents driven by ChR2 expression in DAT-PMV neurons and recorded in VMHVL (inset). Light activation of PMV-DAT neurons expressing ChR2 increased social investigation of a cage mate in DAT-vGlut2 heterozygous animals (control), but not in DAT-vGlut2 knockout animals (n = 6 animals/group; Student’s t test ∗∗∗∗p < 0.0001). (G) Activation did not increase investigation of a familiar object in either group. (H) Pairing one side of a two-chambered box with light stimulation did not cause a significant preference in either group. (I) Social investigation of a resident male by an intruder male was reduced in DAT-vGlut2 knockout animals (n = 6 animals/group; Student’s t test ∗∗∗p < 0.001). (J and K) Social investigation of a male (J) or female (K) intruder by a resident was unaffected. Data are represented as mean ± SEM. See also Figure S4. Cell Reports 2016 16, 304-313DOI: (10.1016/j.celrep.2016.05.067) Copyright © 2016 The Authors Terms and Conditions